4/5/2016 On the road to disease: testing the stress-induced - PDF document

4/5/2016 On the road to disease: testing the stress-induced susceptibility hypothesis in amphibian populations adjacent to roads Hall, E.M., Brunner, J.L, Hutzenbiler, B., Crespi, E.J. School of Biological Sciences, Washington State University, Pullman WA Photo: Alex Shepak Road Map 1. Why stress can increase susceptibility to disease 2. Combining surveillance (eDNA) and dose-response experiments 3. Understanding why variance within a species across an ecological context is important Anthropogenic effects infectious diseases in wildlife 53% (10/19) studies showing an increase in wildlife disease prevalence related to human-modified landscapes Brearley et al. (2012) Biol. Rev. 1

4/5/2016 Anthropogenic effects infectious diseases in wildlife Human modified landscapes Landscape Processes (e.g.): - Continuity and structure - Seasonality and timing - Biogeochemical and physical Transmission Host response + vector/reservoir density • Behavior and migration + inter/intra – specific contact • Life history trade-offs + transport from distant locations Physiological stress response • + patchiness/resource clumping • Disruption of homeostasis (e.g. hormone disruptors) Disease impact Modified from Brearley et al. (2012) Biol. Rev. Stress-induced susceptibility hypothesis Chronic stress is immunosuppressive, therefore environmental change that causes chronic stress will increase susceptibility to infection (Carey et al., 2006 Dev Comp. Immunol) (Rollins-Smith, 2001 Immun Res) Crespi et al, in prep . Dhabhar and McEwen 1997 Brain, Behav, Immun Examples: stress-induced susceptibility in amphibians Belden and Kiesecker 2005 Stressors that suppress immune function: • Glucocorticoids (naturally during metamorphosis) (e.g., Rollins-Smith 2001, Immunol Res ; Belden and Kiesecker 2005, J. Parasitol ; but see Searle et al 2014, J Exp Zool ) • Toxicity (Contaminants, agrochemicals, etc) (e.g., Rohr et al 2008, Nature ; Forson and Storfer 2006, Ecol Appl ) • Nutritional deficit (Gervasi and Foufopoulos 2008, Funct Ecol , Venesky 2012, Oecologia ) Scale up to population level? 2

4/5/2016 Examples: stress-induced susceptibility in amphibians Environmental correlates with disease: • Agricultural fields (e.g., Miller et al 2009) • Urbanization/Industry (e.g., Skelly et al 2006, St. Amour et al 2008) • Catchment position (Gahl and Calhoun 2010) • Roads (Urban 2006, Cons Bio ; Pauza et al 2010, DOA ) Gahl and Calhoun 2010 What is the mechanism? Roads are a major stressor to amphibians Jackson and Jobbagy, PNAS (2005) Hall et al. Unpub Roads are a major stressor to amphibians Calling Vehicle impact e.g., Lengagne 2008, Biol Cons Survival Development rate ? Deformities Susceptibility to mold e.g., Karraker et al 2008, Ecol Appl Growth rate Development rate e.g., Sanzo and Hecnar, 2006, Environ Pollut 3

4/5/2016 Wood frog system - Explosive breeders - Ephemeral fishless ponds - Mortality from ranavirus reaches >90% of tadpoles, symptoms are observable + + : : : + Wood frog system: ranavirus infection across the range Found a higher prevalence of low-level infection in wood frog adults at center of range - No correlation with stress hormones (GCs) - Center of range is also highest density of human population Crespi et al (2015), ICB Testing the stress-induced susceptibility hypothesis 1. Are roads associated with ranavirus die-offs? Host density, behavior, and physiology are affected by roadside conditions, thus ranavirus dynamics will vary by proximity to roads. Approach : Die-off and eDNA surveillance across a forested area dissected by roads 4

4/5/2016 Die-offs were more likely to occur near highways 7 4 5 3 6 9 1 2 8 Hall et al., in prep Wildlife disease surveillance: eDNA eDNA is trace DNA in environmental samples. Mixture of degraded DNA from many different organisms. (Bohmann et al 2014 , Trends Ecol Evol ) Weaknesses Strengths • Not able to distinguish host • Detect multiple species with species infected with one sample (rare & cryptic) pathogen • Relate pathogen and host • High sensitivity prone to densities false positives • Non-invasive, fast and easy • Cannot distinguish between • High sensitivity infectious/degraded pathogens Using eDNA to detect ranaviruses in pond water • Strengths: Not prone to false positives • Weaknesses: Not as sensitive • Some false negatives found -> sample more! Sampled 20 wood frog ponds twice (before and after metamorphic climax) for eDNA (3 filters) and larvae (5 in 12 ponds) + + + + 5

4/5/2016 Using eDNA to detect ranaviruses in pond water RV eDNA titers RV titers peaked reflected tadpole titers around die-off Hall et al., 2015, Molecular Ecology Resources Using eDNA to detect ranaviruses in pond water 2014: Sampled 8 ponds every two weeks: - 3 eDNA samples - 10 larval samples - Stages, densities, water chemistry Monitored for die-offs every week (> 5 carcasses) Hall et al., in prep eggs What’s different about where die -offs occur? Ranaviruses are ubiquitous… O’Connor, K.M., T.A.G. Rittenhouse, and J.L.Brunner, in prep . …so why are die -offs more likely in some ponds but not others? Hall et al ., in prep 6

4/5/2016 Specific hypotheses How might road run-off increase susceptibility to infection? 1. Glucocorticoids may directly down regulate immune function, corticosterone increasing susceptibility. Adults (Hall et al., in prep ) - Roadside had more bloating, >200 m from a road <200 m from a road and bloated adults had hormone profiles indicative of chronic stress Tadpoles (Hall et al., in prep ) - Higher baseline GCs in those collected from roadside ponds (when raised in freshwater or saltwater) Specific hypotheses How might road run-off increase susceptibility to infection? Cl - Cl - 2. Osmoregulation is costly and Cl - reduces the amount of energy Cl - Cl - available for fighting infection. Tadpole energy budget Tadpoles (Hall et al., in prep ) Freshwater Saltwater - Slower growth in roadside ponds with higher salinity - Reduced feeding behavior when raised in road salt Osmoregulation - Gill edema in road salt VS exposure in lab Growth, storage, immune function Specific hypotheses How might road run-off increase susceptibility to infection? 3. Stress of poor conditions alters the timing of developmental traits associated with susceptibility. Tadpoles (Hall et al,. in prep) - Slower development rate in road salt exposure in lab - Greater variation in development rate in roadside ponds Warne et al., 2011, Functional Ecology 7

4/5/2016 Testing the stress-induced susceptibility hypothesis 2 . Do roads increase susceptibility to ranavirus infection? Roadside conditions influence host physiology, thus individual susceptibility to infection will be related to proximity to roads. Approach: Dose-response exposure to FV3 ranavirus Ranavirus (FV3) Dose Response Collect animals from Measure Exposure: susceptibility: ponds varying in Control: 0 pfu proximity to a road • Mortality rate Low: 3x10 3 pfu Avg. stage = 36 • Proportion Med: 3x10 4 pfu infected N=9* High: 3x10 5 pfu N=10 *Collected from ponds that did not have RV die-offs at that time The power of dose-response experiments We can determine both the LD 50 and the distribution of susceptibility using a dose-response experiment Heterogeneous-host Dose response profile Susceptibility distribution model: Predictions Fraction infected/dead Stressed Mean susceptibility Probability density Control Variance parameter Dose Susceptibility Modified from Ben-Ami 2010 Am Nat Variation across and within populations Despite high prevalence of infection across ponds, the response to a (secondary) challenge in the laboratory varied by proximity to a paved road Increasing salinity * * m m m m m m m m m infected Hall et al., in prep 8

4/5/2016 Variation across and within populations Probability tadpoles will Variance in susceptibility to secondary infection succumb to infection Hall et al., in prep Potential explanations for differences in susceptibility • Initial exposure • Mortality to salinity • Susceptible population already succumbed to infection Susceptible Tolerant Potential explanations for differences in susceptibility Roadside experimental enclosures had greater variance in development rate (Hall et al., in prep ), and developmental stages vary in mortality to infection Woodland Roadside Developmental Stage Susceptibility window Time 9